Pyrazolopyrimidines are a general class of compounds which exhibit Aj adenosine receptor affmity. A number of pyrazolo(3,4-d)pyrimidine analogues of isoguanosine and i-methylisoguanosine has been synthesised. All compounds were tested forAi adenosine receptor affinity using a (311) R-PIA competitive binding assay. The N-i and N-5 positions were substituted with a number of different ailcyl and aryi groups. 3-Chiorophenyl substitution of the N-i position and butyl substitution of the N-5 position greatly enhanced the overall adenosine receptor affinity. Substitution by a methyl group at the N-7 position fixed the C-4 position in the imino tautomeric form. This resulted in a marked reduction in activity. The substitution of the N-2 position with a phenyl group produced an analogue with a similar structure to i,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX). A 2-phenyl substituent was favourable for interaction with the adenosine receptor. A number of pyrazolo(3,4-d)pyrirnidine analogues of 4,6-bis-a-carbamoylethylthio-i-phenylthiopyrazolo(3,4-d)pyrinhidine (DJB-KK) has also been synthesised and tested for Aj adenosine receptor affinity. 4,6-Bis-alkylthio-1-phenylpyrazolo(3,4-d)pyrimidines with a-carbamoylethyl and u-carbamoylpropyi groups were compared. The additional methyiene of the a-carbamoylpropyl group produced increased adenosine receptor affinity. 6-a-Carbamoylethylthio-4-mercapto-1-phenylpyrazolo(3,4-d)pyrimidine and 4-cc-carbamoylethylthio- i-phenylpyrazolo(3,4-dlpyrimidine were compared. Substitution of the C-6 position maintained activity, while substitution of the C-4 reduced activity.

The implication of the transmembrane signalling Receptor Tyrosine Kinases (RTKs) in cancer has accelerated the pursuit for drugs to target these molecules. In the process our understanding of how these membrane bound molecules are entangled in cell signalling has significantly expanded. There is now evidence that RTKs can facilitate the formation of a lattice-type network of signalling molecules to elicit whole cell responses to external ligand stimuli. Although beginning to be unravelled, knowledge pertaining to the mechanisms of molecular control that initiate these signalling pathways is still in its infancy. In this thesis, a random mutagenesis approach allowed the identification of the crucial interaction surfaces between membrane-bound EphA3 and its preferential binding partner ephrinA5, that are required to induce the formation of higher-order Eph signalling complexes. Modelling and experimental dissection of this co-ordinated receptor aggregation has provided detailed insights into the molecular mechanisms of Eph receptor activation, which in some aspects may also apply to other members of the RTK family. In particular, the importance of certain molecular interfaces in determining preferential and non-preferential Eph/ephrin interactions, suggests their role in the selection of biologically important binding partners. In addition to the assignment of the ephrin-interaction surfaces, the random mutagenesis strategy also identified a continuous conformational epitope as binding site for an anti-EphA3 monoclonal antibody. Fortuitously, antibody binding to this site functionally mimics ephrin stimulation of EphA3 positive cells, and in particular together with divalent ephrinA5, yields synergistically enhanced EphA3 activation. Elucidation of the underlying mechanism has provided opportunities to develop an efficient EphA3 targeting mechanism that is based on increased affinity and accelerated ephrinA5 uptake as consequence of this unique activation mechanism. On a genetic level, novel oligonucleotide analogues known as Peptide Nucleic Acids (PNAs) were analysed for their ability to sterically inhibit EphA3 DNA transcription and suggest a dosedependent downregulation of EphA3 expression, in malignant melanoma cells. Combined, ephrinA5, the anti-EphA3 MAb (IIIA4) and PNA, offer the possibility to investigate the specific machinery involved in Eph receptor expression and signalling for the specific targeting of EphA3 expressing tumour cells.

The focus of this dissertation was leptin and the leptin receptor, and the role of these genes (OB and OB-R) in the development of obesity and type 2 diabetes in humans and Psammomys obesus, a polygenic rodent model of obesity and type 2 diabetes. Studies in humans showed that circulating leptin concentrations were positively associated with adiposity, and independently associated with circulating insulin and triglyceride concentrations. Analysis of two leptin receptor sequence polymorphisms in a Caucasian Australian population and a population of Nauruan males, with very high prevalence rates of obesity, showed no associations between sequence variation within the OB-R gene and obesity- or diabetes-related phenotypic measures. In addition, these two OB-R polymorphisms were not associated with longitudinal changes in body mass or composition in either of the populations examined. A unique analysis of the effects of multiple gene defects in the Nauruan population, demonstrated that the presence of sequence alterations in both the OB and OB-R genes were associated with insulin resistance. Psammomys obesus is regarded as an excellent rodent model in which to study the development of obesity and type 2 diabetes in humans. Examination of circulating leptin concentrations in Psammomys revealed that, as in humans, leptin concentrations were associated with adiposity, and independently associated with circulating insulin concentrations. This animal model was utilised to examine expression of OB-R, and the regulation of expression of this gene after dietary manipulation. OB-R is known to have several isoforms, and in particular, OB-RA and OB-RB gene expression were examined. OB-RB is the main signalling isoform of the leptin receptors. It has a long intracellular domain and has previously been shown to play an important role in energy balance and body weight regulation in rodents and humans. OB-RA is a much shorter isoform of OB-R, and although it lacks the long intracellular domain necessary to activate the JAK/STAT pathway, OB-RA is also capable of signalling, although to a lesser degree than OB-RB. OB-RA is found to be expressed almost ubiquitously throughout the body, and this isoform may be involved in transport of leptin into the cell, although its role remains unclear. OB-RA and OB-RB were both found to be expressed in a large number of tissues in Psammomys obesus. Interestingly, obese Psammomys were found to have lower levels of expression of OB-RA and OB-RB in the hypothalamus, compared to lean animals. This finding raises the possibility that decreased leptin signalling in the brain of obese, hyperleptinemic Psammomys obesus may contribute to the leptin resistance previously described in this animal model. However, the primary defect is unclear, as alternatively, increased circulating leptin concentrations may lead to down-regulation of leptin receptors. The effect of fasting on leptin concentrations and gene expression of OB-RA and OB-RB was also examined. A 24-hour fast resulted in no change in body weight, but a reduction in circulating leptin concentrations, and an increase in hypothalamic OB-RB gene expression in lean Psammomys. In obese animals, fasting again did not alter body weight, but resulted in an increase in both circulating leptin concentrations and hypothalamic OB-RB gene expression. In the liver, fasting resulted in a large increase in OB-RA gene expression in both lean and obese animals. These results highlighted the fact that regulation of leptin receptor gene expression in polygenic models of obesity and type 2 diabetes is complex, and not solely under the control of circulating leptin concentrations. Sucrose-feeding is an established method of inducing obesity and type 2 diabetes in rodents, and this experimental paradigm was utilised to examine the effects of longer term perturbations of energy balance on the leptin signalling pathway in Psammomys obesus. Addition of a 5% sucrose solution to the diet of lean and obese Psammomys resulted in increased body weight in both groups of animals, however only obese Psammomys showed increased fat mass and the development of type 2 diabetes. The changes in body mass and composition with sucrose-feeding were accompanied by decreased circulating leptin concentrations in both groups of animals, as well as a range of changes in leptin receptor gene expression. Sucrose-feeding increased hypothalamic OB-RB gene expression in obese Psammomys only, while in the liver there was evidence of a reduction in OB-RA and OB-RB gene expression in both lean and obese animals. The direct effects of sucrose on the leptin signalling pathway are unclear, however it is possible to speculate that the effect of sucrose to decrease leptin concentrations may have been involved in the exacerbation of obesity and the development of type 2 diabetes in obese Psammomys, From these studies, it appears that sequence variation in the OB and OB-R genes is unlikely to be a major factor in the etiology of obesity in human populations. The ability to examine regulation of expression of these genes in Psammomys obesus, however, has demonstrated that the effects of nutritional modifications on leptin receptor gene expression need closer attention. The role of the OB and OB-R genes in metabolism and the development of type 2 diabetes also warrants further examination, with particular attention on the differential effects of dietary modifications on leptin receptor gene expression across a range of tissues.

The natriuretic peptides, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) are members of a family of hormones that play an important role in mammalian fluid and electrolyte balance. In the periphery, natriuretic peptides reduce blood volume and subsequently blood pressure by increasing renal natriuresis and diuresis and relaxation of vascular smooth muscle. The actions of natriuretic peptides are mediated via two membrane-linked guanylate cyclase receptors (NPR-GC); natriuretic peptide receptor-A (NPR-A) which has a high affinity for ANP and BNP; and natriuretic peptide receptor-B (NPR-B)which has the greatest affinity for CNP. A third receptor not linked to guanylate cyclase, natriuretic peptide receptor-C (NPR-C) also exists, which binds to ANP, BNP and CNP with a relatively equal affinity, and is involved with clearance of the peptides from the circulation and tissues. The natriuretic peptides are present in the brain and are particularly predominant in cardiovascular and fluid and electrolyte regulating areas such as the anteroventral third ventricle (AV3V) region. This distribution has led to the suggestion natriuretic peptides play a neuromodulatory role in the central control of fluid homeostasis. Natriuretic peptides in the brain have been observed to inhibit the release of other fluid and electrolyte regulating hormones such as arginine vasopressin (AVP) and angiotensin II (AII). Natriuretic peptides have also been identified in the non-mammalian vertebrates although information regarding the distribution of the peptides and their receptors in the non-mammalian brain is limited. In amphibians, immunohistochemical studies have shown that natriuretic peptides are highly concentrated in the preoptic region of the brain, an area believed to be analogous to the A\T3\ region in mammals, which suggests that natriuretic peptides may also be involved in central fluid and electrolyte regulation in amphibians. To date, CNP is the only natriuretic peptide that has been isolated and cloned from the lower vertebrate brain, although studies on the distribution of CNP binding sites in the brain have only been performed in one fish species. Studies on the distribution of ANP binding sites in the lower vertebrate brain are similarly limited and have only been performed in one fish and two amphibian species. Moreover, the nature and distribution of the natriuretic peptide receptors has not been characterised. The current study therefore, used several approaches to investigate the distribution of natriuretic peptides and their receptors in the brain of the amphibian Bufo marinus. The topographical relationship of natriuretic peptides and the fluid and electrolyte regulating hormone arginine vasotocin was also investigated, in order to gain a greater understanding of the role of the natriuretic peptide system in the lower vertebrate brain. Immunohistochemical studies showed natriuretic peptides were distributed throughout the brain and were highly concentrated in the preoptic region and interpeduncular nucleus. No natriuretic peptide-like immunoreactivity (NP-IR) was observed in the pituitary gland. Arginine vasotocin-like immunoreactivity (AvT-IR) was confined to distinct regions, particularly in the preoptic/hypothalamic region and pituitary gland. Double labelling studies of NP-JR and AvT-IR showed the peptides are not colocalised in the same neural pathways. The distribution of natriuretic peptide binding sites using the ligands 125I-rat ANP (125I-rANP) and 125I-porcine CNP (125I-pCNP) showed different distributions in the brain of B. marinus. The specificity of binding was determined by displacement with unlabelled rat ANP, porcine CNP and C-ANF, an NPR-C specific ligand. 125I-rANP binding sites were broadly distributed throughout the brain with the highest concentration in pituitary gland, habenular, medial pallium and olfactory region. Minimal 125I-rANP binding was observed in the preoptic region. Residual 125I-rANP binding in the presence of C-ANF was observed in the olfactory region, habenular and pituitary gland indicating the presence of both NPR-GC and NPR-C in these regions. 125I-pCNP binding was limited to the olfactory region, pallium and posterior pituitary gland. All 125I-pCNP binding was displaced by C-ANF which suggests that CNP in the brain of B. marinus binds only to NPR-C. Affinity cross-linking and SDS-PAGB demonstrated two binding sites at 136 kDa and 65 kDa under reducing conditions. Guanylate cyclase assays showed 0.1 µM ANP increased cGMP levels 50% above basal whilst a 10-fold higher concentration of CNP was required to produce the same result. Molecular cloning studies revealed a 669 base pair fragment showing 91% homology with human and rat NPR-A and 89% homology with human, rat and eel NPR-B. A 432 base pair fragment showing 67% homology to the mammalian NPR-C and 58% homology with eel NPR-D was also obtained. The results show natriuretic peptides and their receptors are distributed throughout the brain of B. marinus which indicates that natriuretic peptides may participate in a range of regulatory functions throughout the brain. The potential for natriuretic peptides to regulate the release of the fluid and electrolyte regulating hormone AVT also exists due to the high number of natriuretic peptide binding sites in the posterior pituitary gland. At least two populations of natriuretic peptide receptors are present in the brain of B. marinus, one linked to guanylate cyclase and one resembling the mammalian clearance receptor. Furthermore, autoradiography and guanylate cyclase studies suggest ANP may be the major ligand in the brain of B. marinus, even though CNP is the only natriuretic peptide that has been isolated from the lower vertebrate brain to date.

8-Cyclopentyl-3-(3-(4-fluorosulfonylbenzoyl)oxy)propyl-propylxanthine (44, FSCPX) has been reported to exhibit potent and selective irreversible antagonism of the A1 adenosine receptor when using in vitro biological preparations. However, FSCPX (44) suffers from cleavage of the ester linkage separating the reactive 4-(fluorosulfonyl)phenyl moiety from the xanthine pharmacophore when used in in vivo biological preparations or preparations containing significant enzyme activity, presumably by esterases. Cleavage of the ester linkage renders FSCPX (44) inactive in terms of irreversible receptor binding. In order to obtain an irreversible A1 adenosine receptor antagonist with improved stability, and to further elucidate the effects of linker structure on pharmacological characteristics, several FSCPX (44) analogues incorporating the chemoreactive 4-(fluorosulfonyl)phenyl moiety were targeted, where the labile ester linkage has been replaced by more stable functionalites. In particular, ether, alkyl, amide and ketone linkers were targeted, where the length of the alkyl chain was varied from between one to five atoms. Synthesis of the target compounds was achieved via direct attachment of the N-3 substituent to the xanthine. These compounds were then tested for their biological activity at the A1 adenosine receptor via their ability to irreversibly antagonise the binding of [3H]-8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX, ( 9) to the A1 adenosine receptor of DDT1 MF-2 cells. For comparison, the xanthines were also tested for their ability to inhibit the binding of [3H]-4-(2-[7-amino-2-{furyl} {1,2,4}- triazolo{2,3-a} {1,3,5}triazin-5-ylamino-ethyl)]phenol ([3H]ZM241385, 36) to the A2A adenosine receptor of PC-12 cells. The results suggest that the length and chemical composition of the linker separating the reactive 4-(fluorosulfonyl)phenyl moiety from the xanthine ring contribute to the potency and efficacy of the irreversible A1 adenosine receptor ligands. Like FSCPX (44, IC50 A1 = 11.8 nM), all derivatives possessed IC50 values in the low nM range under in vitro conditions. Compounds 94 (IC50 A1 = 165 nM), 95 (IC50 A1 = 112 nM) and 96 (IC50 A1 = 101 nM) possessing one, three and five methylene spacers within the linkage respectively, exhibited potent and selective binding to the A1 adenosine receptor versus the A2A adenosine receptor. Compound 94 did not exhibit any irreversible binding at A1 adenosine receptors, while 95 and 96 exhibit only weak irreversible binding at A1 adenosine receptors. Those compounds containing a benzylic carbonyl separating the 4-(fluorosulfonyl)phenyl moiety from the xanthine ring in the form of an amide (119, IC50 A1 = 24.9 nM, and 120, IC50 A1 = 21 nM) or ketone (151, IC50 A1 = 14 nM) proved to be the most potent, with compound 120 exhibiting the highest selectivity of 132-fold for the A receptor over the A2A receptor. compounds 119, 120 and 151 also strongly inhibited the binding of [3H]DPCPX irreversibly (82%, 83% and 78% loss of [3H]DPCPX binding at 100 nM respectively). compounds 120 and 151 are currently being evaluated for use in in vivo studies. Structure-activity studies suggest that altering the 8-cycloalkyl group of A1 selective xanthines for a 3-substituted or 2,3-disubstituted styryl, combined with N-7 methyl substitution will produce a compound with high affinity and selectivity for the A2A adenosine receptor over the A1 adenosine receptor. Compound 167 (IC50 A2A = 264 nM) possessing 8-(m-chloro)styryl substitution and the reactive 4-(fluorosulfonyl)phenyl moiety separated from the xanthine ring via an amide linker in the 3-position (as for 119 and 120), exhibited relatively potent binding to the A2A adenosine receptor of PC-12 cells, with a 16-fold selectivity for that receptor over the A1 adenosine receptor. However, compound 167 exhibited only very weak irreversible binding at A2A adenosine receptors. Overall, at this stage of biological testing, compound 120 appears to possess the most advantageous characteristics as an irreversible antagonist for the A1 adenosine receptor. This can be attributed to its high selectivity for the A1 adenosine receptor as compared to the A2A adenosine receptor. It also has relatively high potency for the A1 adenosine receptor, a concentration-dependent and selective inactivation of A1 adenosine receptors, and unbound ligand is easily removed (washed out) from biological membranes. These characteristics mean compound 151 has the potential to be a useful tool for the further study of the structure and function of the A1 adenosine receptor.

Novel approaches to cancer treatment in the context of Gene Therapy have been gaining popularity as an alternative to conventional therapies which have proven to lack specificity, resulting in tumour cell resistance, tumour progression and mortality. As a consequence the use of adenoviruses has been widely developed not only as a replication deficient vector for gene therapy but also as a replication competent oncolytic agent designed to selectively target and kill tumour cells. Unfortunately their success in clinical application has been limited, and it has been suggested that a lack of the primary viral attachment receptor 'CAR' could be a barrier to infection by limiting access to target cells. If Ad/CAR binding is the rate limiting step for successful Ad therapy, it is essential to establish a CAR expression profile in normal and tumour tissue, and in tumour progression, to enable more effective targeted therapy. Furthermore, in the context of using adenovirus as an anticancer strategy by exploiting its replicative lysis, it is important to explore whether Ad success is affected by CAR expression and to identify factors downstream of CAR that may be influential in this process. In the first experimental chapter, an in vivo immunohistochemical analysis of tissue array slides determined CAR expression in a range of normal and tumour tissue. CAR was differentially expressed dependent on cell of origin, with normal stem cells and basal cells displaying very high CAR, signifying its importance in early development and differentiation. Epithelial cells were also high in CAR but its expression was negligible in mesenchymal, lymphoid and neural cells. This trend was also reflected in most tumour tissue albeit with a general decrease in CAR compared to corresponding normal tissue of the same organ. An exception was the blastic tumours which displayed high CAR reflecting their embryonic state of derivation. CAR expression also decreased in high grade, poorly differentiated tumours of the prostate, stomach and breast compared to their well differentiated counterparts. In the second experimental chapter, a more comprehensive study of breast cancer biopsy specimens was undertaken, to determine both the expression of CAR and the tumour suppressor gene p53 in relation to tumour grade. The rationale being that both loss of CAR expression and p53 mutation (resulting in loss of function), have been associated with tumour progression. It is possible that CAR and p53 interact directly or indirectly and may be modulated by each other. This study revealed a decrease in both CAR and hormone receptor expression and an increase in p53 'mutational' status with increasing tumour grade. These three factors when compared independently to tumour grade are statistically significant associations, implying that CAR expression and hormone responsiveness decrease with tumour progression and p53 function is compromised or lost via mutation. There was also a significant association between CAR expression and hormone receptor status, however a significant association between CAR expression and p53 status within the tumour grades was not found. Treatment outcome with Ads will also depend on defining factors downstream of CAR attachment that affect adenovirus 'permissivity', which is ultimately measured by viral replication and cell death, relying on the bystander effect to eradicate all tumour cells. The in vitro analysis revealed statistically significant associations between CAR receptor expression, 'infectivity' (virus infection) and permissivity. Cell lines that were more susceptible to Ad5 were generally of epithelial origin, had high CAR, and were easily infected. However there were exceptions and CAR was not the sole determinant in adenovirus cell entry nor in its ability to replicate and kill the cell. Permissivity was also related to p53 status. Thus, although CAR expression may indeed be a limiting factor, it is apparent that a combination of other events contributes to a deficient infection, especially in the deregulated tumour environment. The results presented in this thesis clearly demonstrate that there is more to the story of 'CAR' which hints that its role in viro-oncotherapy is not limited solely to its function as an attachment receptor for adenovirus but may also involve its function as a cell adhesion molecule and signal transducer. The further elucidation of these aspects of CAR�s potential role in the scheme of tumour biology may alter the course and strategy of cancer therapy in the future.

The neurohypophysial peptide system is involved in modulating a variety of physiological, neurological, and behavioral responses in vertebrates. The principal forms of these peptides in non-mammalian tetrapods are vasotocin (VT) and mesotocin (MT). The studies described in this thesis used pharmacological, molecular, and biochemical techniques, along with phylogenetic analyses, to identify and characterize the mRNA sequences encoding the neurohypophysial peptide precursor proteins and their receptors in urodele amphibians. The cDNAs encoding preproVT and preproMT were amplified by PCR from the brains of two salamander species; the rough-skinned newt, Taricha granulosa, and the red-legged salamander, Plethodon shermani. The neurohypophysial peptides encoded by the identified Taricha cDNAs were VT and MT; the Plethodon cDNAs encoded VT and a novel MT-like peptide, [Val⁴]-MT. Phylogenetic analyses grouped both the Taricha and Plethodon preproVT and preproMT-like sequences with previously identified tetrapod preproVT-like and preproMT-like sequences, respectively. Additional analysis of the preproneurohypophysial sequences indicated that gene conversion (non-homologous crossing over of DNA sequences) appears to have occurred more frequently in mammals than in other tetrapod classes. The cDNAs encoding the VT receptor (VTR) and MT receptor (MTR) were amplified from the brain of T. granulosa by PCR. Sequence identity, and phylogenetic analysis, indicated that the Taricha MTR and VTR were most similar to MTR/OTRs and V[subscript 1a]-like VTRs, respectively. Distribution of PCR amplicons specific to the Taricha MTR and VTR matched previously reported tissue distributions of MTRs and VTRs in other vertebrates in every tissue but kidney, from which the Taricha primers were unable to amplify a cDNA product. Binding experiments of transiently expressed Taricha MTR indicated two binding states, and allowed the determination of ligand binding affinities for this receptor. Inositol phosphate accumulation assays demonstrated that the expressed Taricha MTR and VTR cDNA produced functional receptors, and allowed calculation of ligand potencies of activation and inhibition. Surprisingly, an antagonist frequently used in behavioral experiments to specifically block VTR activity, inhibited inositol phosphate accumulation in cells transfected with either the Taricha MTR or VTR. In conclusion, these studies report the first identified cDNA sequences encoding the preproVT, preproMT, MTR, and V[subscript 1a]-like VTR proteins from urodele amphibians. / Graduation date: 2005

The main objectives of the current study were to obtain the complete cDNA sequence of an opioid-like receptor from an amphibian, the roughskin newt, Taricha granulosa, and investigate the receptor's tissue distribution and regulation following chronic exposure to the glucocorticoid corticosterone (CORT). Degenerate primers designed in highly conserved regions of characterized opioid receptors were used to amplify opioid-like receptor fragments from a newt brain cDNA library. Receptor fragments with high sequence identity to the orphanin opioid receptor type, also termed the 'opioid receptor-like' (ORL1) receptor, were selected for 3' and 5' RACE (rapid amplification of cDNA ends) reactions to obtain the full-length receptor cDNA sequence. By this approach, we obtained a cDNA sequence that putatively encodes a 368 amino acid protein with high sequence identity (57%) to the human ORL1 receptor. Therefore, hereafter we refer to this receptor as the newt ORL1-like (nORL) receptor. The nORL receptor also has identity with the mammalian kappa (K) opioid receptor at a number of residues that may enable it to recognize both ORL1- and K- receptor selective ligands. The tissue distribution of the nORL receptor was determined by reverse-transcriptase polymerase chain reaction (PCR). RNA from a variety of tissues was reverse-transcribed into cDNA using an oligo-dT primer, and the resultant cDNA was used as template in PCR reactions with nORL receptor-specific primers. Appropriately sized amplicons were produced in reactions with cDNA template originating from newt brain, spinal cord, and lungs. No amplification occurred in reactions with template cDNA from newt spleen, small intestine, heart, liver, sperm duct, bladder, or kidney. The regulation of the nORL receptor following chronic exposure to the glucocorticoid corticosterone was investigated using real-time PCR. Animals were exposed continuously to CORT for 10 days using surgically implanted Silastic capsules packed with CORT powder. Control animals received empty Silastic capsules, or no treatment. The relative quantitation of the nORL receptor messenger RNA (mRNA) was achieved by real-time PCR, and mRNA levels for the hormone-treated animals were compared to those of the controls. The same samples were used for the relative quantitation of intracellular glucocorticoid receptor (iGR) mRNA. There was no change in the expression of mRNA for the nORL receptor or the iGR following chronic exposure to CORT as compared to the controls. In conclusion, this study provides evidence for an opioid-like receptor in the roughskin newt that has high sequence identity to the mammalian ORL1 opioid receptor. To the best of our knowledge, this is the first complete opioid receptor cDNA sequence obtained for an amphibian. The nORL receptor appears to principally function in central nervous system (CNS) processes in the newt, as evidenced by its primary localization to brain and spinal cord. The role of the nORL receptor in the periphery may be limited to a function in the lungs, and awaits further investigation. The current study was also the first to investigate the effects of a stress hormone on the regulation of an opioid receptor in an amphibian. Our results indicate that chronic exposure to the stress hormone corticosterone does not impact the levels of nORL receptor or intracellular glucocorticoid receptor mRNA in the newt spinal cord. / Graduation date: 2003

In addition to the well-characterized genomic mechanism of steroid action that uses intracellular receptors, steroid hormones also signal through nongenomic processes that use membrane receptors. A membrane receptor for corticosterone (CORT) has been described in brains of the roughskin newt (Taricha granulosa). This receptor is believed to be a G-protein coupled receptor because corticosterone binding is inhibited by guanyl nucleotides and enhanced by Mg�����. The studies described in this thesis use biochemical, pharmacological and molecular techniques to characterize the newt neuronal membrane glucocorticoid receptor (mGR) in pursuit of its molecular identification. The mGR was successfully solubilized from newt neuronal membranes and conditions were defined that maintained corticosterone binding activity for further study. The solubilized receptor was partially purified using standard chromatographic techniques and an immobilized ligand affinity resin (CORT-Sepharose). These chromatographic studies were combined with the use of a novel photoaffinity ligand (azido-CORT) to biochemically characterize the mGR protein, finding that it is an acidic glycoprotein with an apparent molecular weight of 63 kDa and an isoelectric point of approximately 5.0. Pharmacological studies with mGR showed that a subset of kappa opioid ligands displaced corticosterone from the receptor binding site with K[subscript i] values in the nanomolar to low-micromolar range. The interaction of mGR with kappa opioid ligands was specific because no mu-, delta-, or orphanin-specific opioid ligands were effective at displacing corticosterone from the receptor. These data suggest that the newt neuronal mGR may be a kappa-opioid like receptor. Finally, molecular studies were used to clone a novel newt brain protein, neuronal axonal protein 22 (NAP-22), that was identified in a protein differential display strategy designed to identify mGR. Studies with the cloned and expressed NAP-22 protein suggest that it is not the mGR but, instead, may be a mGR-associated protein. These studies provided new information about the biochemical and pharmacological properties of mGR, and may have discovered a protein that is associated with the newt neuronal mGR. / Graduation date: 1999

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 127-155).